Our laboratory has identified the T-box transcription factor brachyury as a driver of the epithelial-mesenchymal phenotypic transition (EMT) of human carcinomas. We have demonstrated that high levels of expression of brachyury drive carcinoma cells into a mesenchymal-like phenotype, inducing up-regulation and down-regulation of mesenchymal and epithelial proteins, respectively. Brachyury was also shown to increase tumor cell motility and invasiveness in vitro and to enhance the tumor's ability to disseminate in vivo in animal models of cancer. In addition to promoting tumor dissemination, we have now shown that brachyury expression induces tumor resistance to a variety of cancer therapies, including chemotherapy, radiation, and small-molecule targeted therapies. To investigate the expression of brachyury protein in various types of cancer, our laboratory has now developed and thoroughly characterized a novel rabbit monoclonal antibody (MAb 54-1) that reacts with high affinity and specificity with at least two isoforms of human brachyury. We demonstrated that brachyury protein is expressed both in primary and metastatic lesions of lung and breast cancer. This pattern of expression in tumors contrasted with the expression of brachyury in normal tissues, which was limited to normal testis, some thyroid tissues and a subpopulation of normal B cells. Utilizing the MAb 54-1 antibody we have now characterized the pattern of brachyury nuclear vs. cytosolic expression in a range of cancer types, including in chordoma, embryonal carcinomas, and small cell lung carcinomas. These studies provided a rationale for the use of vaccine approaches targeting brachyury for the therapy of several types of human tumors. Currently, two vaccine platforms, a recombinant yeast-brachyury vaccine and a poxviral vector-based brachyury vaccine are undergoing Phase I and II clinical trials in patients with advanced carcinomas or chordoma. As tumors are typically heterogeneous in terms of expression of different tumor-associated antigens (TAAs), we have now developed a novel cancer vaccine platform consisting of a mixture of three recombinant adenoviruses (termed Tri-Ad5) encoding for brachyury, mucin-1 (MUC1) and carcinoembryonic antigen (CEA). We have shown the the Tri-Ad5 vaccine is able to activate antigen-specific human T cells directed against all three antigens in vitro and in vaccinated mice. It is expected that an immunotherapeutic vaccine regimen targeting three distinct TAAs would be therapeutically advantageous over the use of a vaccine targeting a single antigen. A signaling pathway that is frequently deregulated in human carcinomas and has been explored as a therapeutic target involves the activation of the epidermal growth factor receptor (EGFR). Inhibition of EGFR via the small-molecule inhibitors erlotinib and gefitinib commonly results in tumor resistance, even in patients with EGFR-mutant tumors that initially show substantial clinical responses. We have now demonstrated that generation of erlotinib-resistant lung cancer cells in vitro results in phenotypic alterations reminiscent of an EMT, concomitant with a robust upregulation of the IL-8/IL-8R axis. We have also shown that blockade of IL-8 signaling effectively reduces mesenchymal features of the resistant cells and also markedly enhances their susceptibility to erlotinib. These results provided the rationale for the development of new therapeutic approaches involving blockade of IL-8 signaling for the management of acquired resistance to EGFR inhibition in patients with lung cancer. In additional studies we also demonstrated that EMT can significantly reduce the susceptibility of cancer cells to lysis by both antigen-specific T cells and natural killer (NK) cells. In search for mechanisms of immunotherapy resistance of tumor cells undergoing EMT, we have now discovered a positive association between brachyury and MUC1, a tumor antigen overexpressed in the majority of carcinomas, which mediates oncogenic signaling and confers resistance to genotoxic agents. We found that MUC1 is concomitantly upregulated in tumor cell lines that highly express brachyury, a positive association that was also observed with patient-derived tumor tissues. Inhibition of MUC1 by siRNA-based gene silencing markedly enhanced the susceptibility of brachyury-expressing cancer cells to killing by immune cytotoxic cells. These studies indicated a protective role for MUC1 in brachyury-expressing cancer cells, and suggested that inhibition of MUC1 can restore the susceptibility of mesenchymal-like cancer cells to immune attack. We have recently conducted studies to identify and potentially repurpose FDA-approved compounds capable of reducing mesenchymal features of human lung carcinoma cells, which could be used in combination with immunotherapies or chemotherapeutic strategies to improve clinical responses. In collaboration with NCTAS (NIH), we have utilized a quantitative high throughput screening (qHTS) assay of a pharmaceutical collection of more than 2,000 compounds and identified the estrogen receptor antagonist, fulvestrant, as able to reduce mesenchymal features of lung carcinoma cells, resulting in tumor sensitization to the cytotoxic effect of antigen-specific T cells, natural killer (NK) effectors cells and chemotherapy both in vivo and in vitro. To our knowledge, this was the first report defining a potential role for estrogenic signaling in promoting tumor resistance to immune-mediated cytotoxicity and chemotherapy in lung cancer. Our data also supports further investigations on the use of fulvestrant in combination with chemotherapy or immunotherapy for the treatment of lung cancer.